Over the last three decades, the three-dimensional structures of a variety of biologically interesting macromolecular complexes with helical, icosahedral, octahedral or no symmetry have been determined using images recorded with an electron microscope. High resolution electron microscopic analyses of two-dimensional crystals have in a few instances resulted in structures of proteins with atomic resolution. While analysis of non-crystalline “single particle” suspensions has resulted in the determination of many structures at medium (7 Å-15 Å) resolutions.
Although the last decade has seen considerable improvements in the speed of image analysis, the availability of sufficient numbers of high quality images continues to be the rate limiting step for almost all biological structure determination projects.
An obvious approach to increase the throughput in structure determination efforts is to introduce automated data collection routines that emulate the manual data recording strategies employed by experienced users. Several reports have already appeared in the literature with this aim in mind, and have been pioneered by researchers interested in structure determination using electron tomography, single molecule and helical reconstruction and two-dimensional crystallography. The availability of the partially computerized customized microscopy (“CM”) series of microscopes which allowed the construction of efficient hardware and software interfaces to access key microscope controls has been an important element in the success of these efforts. A few recent reports have reported the introduction of significant levels of automation in the data collection process using CM series microscopes, however, these microscopes are no longer manufactured.
For example, Carragher et al., disclose partially automated methods for obtaining electron micrographs in “Leginon: An Automated System for Acquisition of Images from Vitreous Ice Specimens,” Journal of Structural Biology, (132, 33-45 (2000)). Similarly, Fung discloses processes for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles in “Toward Fully Automated High-Resolution Electron Tomography,” Journal of Structural Biology, (116, 181-189 (1996)). Fung also describes systems that automate the various steps necessary for data collection in tomography. Kisseberth et al. disclose tools that may be used in the control of a remotely situated electron microscope in “emScope: A Tool Kit for Control and Automation of a Remote Electron Microscope”, Journal of Structural Biology (120, 309-319(1997)).
However, much further work is needed in order to eliminate interfaces that decrease image clarity, to allow for more efficient data collection, and to decrease the need for operator interface with the microscope.